(A) Chromogranin A (CHGA) polymorphisms in individuals from population blood pressure (BP) extremes: haplotype sliding-window analysis. Four common variants (each minor allele frequency 8%) (Table 1) were scored to span the CHGA locus. The exon/intron structure of the locus, as well as the positions for all common single nucleotide polymorphisms (SNPs) (21), is depicted in the schematic at the bottom. Results for haplotypes composed of 1, 2, 3, or 4 SNPs were computed by the SNP expectation maximization algorithm for the dichotomous BP trait, and significance is plotted as reciprocal p values for each group: all subjects (left), men only (center), and women only (right). The p values were derived from omnibus permutation tests. Significant (<0.05) p values are shown at the appropriate point. (B) CHGA common extended haplotype GGCC: sex-dependent effect on BP as a quantitative trait in population BP extremes. Extended 4-SNP haplotype GGCC is the most common haplotype in this population (at 57.4% of chromosomes; see inset). The effect of haplotype GGCC on the quantitative trait diastolic blood pressure (DBP) is illustrated separately for men and women. There is a significant overall effect for genotype (p = 0.023), as well as an effect in men alone (p = 0.006).

(A) Hypertension, sex, and CHGA secretion. Resting plasma concentration of the CHGA precursor (epitope: CHGA _116-439) was measured in plasma from seated human subjects with normal renal function (serum creatinine 1.5 mg/dl). We studied subjects with a diagnosis of essential hypertension (HT), versus unmedicated control subjects (NT) with normal blood pressure (BP). (B) Hypertension, sex, and catecholamine secretion. The same individuals were studied for plasma norepinephrine concentration (CHGA and norepinephrine were measured in the same plasma sample). (C) CHGA as a predictor of catecholamine secretion. Individuals were divided into quantiles above and below the median value for plasma CHGA concentration, and plasma catecholamine concentrations were calculated in the 2 groups (CHGA and catecholamine were measured in the same plasma sample). (D) CHGA 3'-untranslated region (3'-UTR) variant C+87T: influence on circulating CHGA. Resting plasma concentration of the CHGA precursor (epitope: CHGA _116-439) was measured in plasma from 578 genotyped white subjects (187 men, 391 women). Each subject had normal renal function (serum creatinine 1.5 mg/dl). Since the distribution of plasma CHGA _116-439 concentration in this sample deviated substantially from normality (skewness = 6.03 ± 0.10, kurtosis = 55.2 ± 0.20), we used a nonparametric median test (evaluating whether 2 or more independent samples [defined by diploid genotype] are drawn from populations with the same median, using the chi-square statistic). There was no gene-by-sex interaction on the CHGA trait (p = 0.57). HWE = Hardy-Weinberg equilibrium; other abbreviations as in Figure 1.

Provocation of efferent sympathetic outflow was undertaken in each subject by immersion of 1 hand in ice water (at 0°C) for 1 min, with continuous blood pressure (BP) monitoring. Results are shown for final (post-cold) systolic blood pressure (SBP), and analyzed by generalized estimating equations, establishing an exchangeable correlation matrix to take into account intratwin-pair correlations. (A) CHGA C+87T genotype effect on post-stress SBP; data are shown in all persons, as well as men and women separately. Genotype affected the trait (p = 0.042), and there was no gene-by-sex interaction (p = 0.159) on trait. (B) Sex effect on post-stress SBP (p = 0.009). 3'-UTR = 3'-untranslated region.

(A) Sequence alignment of the CHGA 3'-untranslated region (3'-UTR) across different species. 3'-UTR sequences are aligned across 7 species, to illustrate conservation at positions of 3 naturally occurring human polymorphisms: C+87T (C11825T), C+96T (C11834T), and C+274T (C12012T). (B) Human CHGA naturally occurring 3'-UTR variants: effects on transfected reporter gene expression in chromaffin cells. The 3'-UTR variants tested were: C+87T (C11825T), C+96T (C11834T), and C+274T (C12012T). Results of luciferase activity measurements 24 h after transfection of the 4 versions of the 407-bp CHGA 3'-UTR (wild type [W-T] and 3 variants) into chromaffin cells. Results are expressed as the ratio of firefly luciferase/Renilla luciferase (encoded by the transfection efficiency plasmid pRL-TK). Each experiment was performed in triplicate, and such experiments were repeated at least twice. The W-T contains the major (C) allele at all 3 positions. Variant constructs contain the minor (T) allele at the indicated position, but the major (C) at the 2 other positions.

Effect of small interfering RNA (siRNA) to "silence" expression of CHGA protein in sympathoadrenal PC12 cells. PC12 cells were grown after transfection with the indicated amount of 22-bp siRNA-rat CHGA duplexes at 4 µg/well for 72 h. (A) Visualization of CHGA immunoreactivity by chemiluminescent immunoblot. Whole cell lysates were prepared, and expression of CHGA and actin (control, "housekeeping" protein) was evaluated by SDS-PAGE followed by immunoblotting using a rabbit polyclonal anticatestatin (rat CHGA _367-387) antibody or a monoclonal antiactin primary antibody. (B) Densitometry to quantify CHGA and actin protein expression, using NIH image v1.6 software. (C and D) Electron microscopy. Ultrastructural examination of the effect of siRNA CHGA "silencing" on dense-core granule biogenesis in sympathoadrenal PC12 cells. Cells were grown in the presence of 22-bp siRNA-rat CHGA duplexes (D), or mock/control (C) at 4 µg/well for 72 h. Aldehyde-fixed cells were processed for electron microscopy as described in the Methods section. Dense core granules (arrowheads) are seen either "docked" to, or in the vicinity of the plasma membrane. Note that fewer secretory granules are present in the cytoplasm of siRNA-rat CHGA-treated cells. (C) Control (mock-treated) PC12 cells. (D) CHGA siRNA-treated PC12 cells. Scale bar: 500 nm. (E) Quantification of the abundance of dense-core secretory granules reveals a decreased number of granules per cell planes, as defined by the number of granules found in an XY section of the midcell body. n = 40 for both populations of cells. ***p < 0.0001, by t test. er = endoplasmic reticulum; m = mitochondria; n = nucleus.